Structural and functional diversity of adaptor proteins involved in tyrosine kinase signalling

Adaptors are proteins of multi-modular structure without enzymatic activity. Their capacity to organise large, temporary protein complexes by linking proteins together in a regulated and selective fashion makes them of outstanding importance in the establishment and maintenance of specificity and efficiency in all known signal transduction pathways. This review focuses on the structural and functional characterisation of adaptors involved in tyrosine kinase (TK) signalling. TK-linked adaptors can be distinguished by their domain composition and binding specificities. However, such structural classifications have proven inadequate as indicators of functional roles. A better way to understand the logic of signalling networks might be to look at functional aspects of adaptor proteins such as signalling specificity, negative versus positive contribution to signal propagation, or their position in the signalling hierarchy. All of these functions are dynamic, suggesting that adaptors have important regulatory roles rather than acting only as stable linkers in signal transduction.     

A perspective on non-catalytic Src homology (SH) adaptor signalling proteins

Intracellular adaptor signalling proteins are members of a large family of mediators crucial for signal transduction pathways. Structurally, these molecules contain one Src Homology 2 (SH2) domain and one or more Src Homology 3 (SH3) domain(s); with either a catalytic subunit, or with other non-catalytic modular subunits. Cells depend on these regulatory signalling molecules to transmit information to the nucleus from both external and internal cues including growth factors, cytokines and steroids. Although there is a vast library of adaptor signalling proteins expressed ubiquitously in cells, the vital role these SH containing proteins play in regulating cellular signalling lacks the recognition they deserve. Their target selection method via the SH domains is simple yet highly effective. The SH3 domain(s) interact with proteins that contain proline-rich motifs, whereas the SH2 domain only binds to proteins containing phosphotyrosine residues. This unique characteristic physically enables proteins from a diverse range of networks to assemble for amplification of a signalling event. The biological consequence generated from these adaptor signalling proteins in a constantly changing microenvironment have profound regulatory effect on cell fate decision particularly when this is involved in the progression of a diseased state.     

How regulators of G protein signaling achieve selective regulation

The regulators of G protein signaling (RGS) are a family of cellular proteins that play an essential regulatory role in G protein-mediated signal transduction. There are multiple RGS subfamilies consisting of over 20 different RGS proteins. They are basically the guanosine triphosphatase (GTPase)-accelerating proteins that specifically interact with G protein alpha subunits. RGS proteins display remarkable selectivity and specificity in their regulation of receptors, ion channels, and other G protein-mediated physiological events. The molecular and cellular mechanisms underlying such selectivity are complex and cooperate at many different levels. Recent research data have provided strong evidence that the spatiotemporal-specific expression of RGS proteins and their target components, as well as the specific protein-protein recognition and interaction through their characteristic structural domains and functional motifs, are determinants for RGS selectivity and specificity. Other molecular mechanisms, such as alternative splicing and scaffold proteins, also significantly contribute to RGS selectivity. To pursue a thorough understanding of the mechanisms of RGS selective regulation will be of great significance for the advancement of our knowledge of molecular and cellular signal transduction.     

A systems biology perspective on protein structural dynamics and signal transduction

The functional dynamics of signal transduction through protein interaction networks are determined both by network topology and by the signal processing properties of component proteins. In order to understand the emergent properties of signal transduction networks in terms of information processing, storage and decision making, we not only need to map the so-called 'interactome' but, perhaps more importantly, we also have to understand how the structural dynamics of constituent proteins shape non-linear responses through cooperativity and allostery. Several in silico methods have been developed to identify networks of cooperative residues in proteins and help infer their mode of action. Applying this type of analysis to important classes of modular signal transduction domains should, in principle, allow the function of these proteins to be abstracted in terms of their information processing characteristics, permitting better comprehension of the systemic properties of biological networks.     

Minimal requirements for oxygen sensing by the aerotaxis receptor Aer

The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra-subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full-length and truncated monomer. Either the PAS or signalling domains could be deleted from the non-signalling subunit of the heterodimer, but removing 16 residues from the C-terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared.     

Dystrophin complex functions as a scaffold for signalling proteins

Dystrophin is a 427 kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

An investigation of spatial signal transduction in cellular networks

ABSTRACT: BACKGROUND: Spatial signal transduction plays a vital role in many intracellular processes such as eukaryotic chemotaxis, polarity generation, cell division. Furthermore it is being increasingly realized that the spatial dimension to signalling may play an important role in other apparently purely temporal signal transduction processes. It is being recognized that a conceptual basis for studying spatial signal transduction in signalling networks is necessary. RESULTS: In this work we examine spatial signal transduction in a series of standard motifs/networks. These networks include coherent and incoherent feedforward, positive and negative feedback, cyclic motifs, monostable switches, bistable switches and negative feedback oscillators. In all these cases, the driving signal has spatial variation. For each network we consider two cases, one where all elements are essentially non diffusible, and the other where one of the network elements may be highly diffusible. A careful analysis of steady state signal transduction provides many insights into the behaviour of all these modules. While in the non-diffusible case for the most part, spatial signalling reflects the temporal signalling behaviour, in the diffusible cases, we see significant differences between spatial and temporal signalling characteristics. Our results demonstrate that the presence of diffusible elements in the networks provides important constraints and capabilities for signalling. CONCLUSIONS: Our results provide a systematic basis for understanding spatial signalling in networks and the role of diffusible elements therein. This provides many insights into the signal transduction capabilities and constraints in such networks and suggests ways in which cellular signalling and information processing is organized to conform to or bypass those constraints. It also provides a framework for starting to understand the organization and regulation of spatial signal transduction in individual processes.     

Signalling pathways in two-component phosphorelay systems

Two-component systems are characterized by phosphotransfer reactions involving histidine and aspartate residues in highly conserved signalling domains. Although the basic principles of signal transduction by these systems have been elucidated, several important aspects, such as their integration into more complex cellular regulatory networks and the molecular basis of the specificity of signal transduction, remain unknown.     

A highly sensitive near‐infrared fluorescent detection method to analyze signalling pathways by reverse‐phase protein array

The comprehensive and quantitative analysis of the protein phosphorylation patterns in different cellular context is of considerable and general interest. The ability to quantify phosphorylation of discrete signalling proteins in large collections of biological samples would greatly favour the development of systems biology in the field of cell signalling. Reverse‐phase protein array (RPPA) potentially represents a very attractive approach to map signal transduction networks with high throughput. In the present report, we describe an improved detection method for RPPA combining near‐infrared with one or two rounds of tyramide‐based signal amplification. The LOQ was lowered from 6.84 attomoles with a direct detection protocol to 0.21 attomole with two amplification steps. We validated this method in the context of intracellular signal transduction triggered by follicle‐stimulating hormone in HEK293 cells. We consistently detected phosphorylated proteins in the sub‐attomole range from less than 1 ng of total cell extracts. Importantly, the method correlated with Western blot analysis of the same samples while displaying excellent intra‐ and inter‐slide reproducibility. We conclude that RPPA combined with amplified near‐infrared detection can be used to capture the subtle regulations intrinsic to signalling network dynamics at an unprecedented throughput, from minute amounts of biological samples.     

Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions

Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, which catalyse downstream reactions. The concept of raft lipid-based membrane domains provides a different principle for compartmentalization and segregation of membrane constituents. Accordingly, rafts are defined by the physical properties of the lipid bilayer and function by selective partitioning of membrane lipids and proteins into membrane domains of specific phase behaviour and lipid packing. Here, I will discuss the interplay of these independent principles of protein scaffolds and raft lipid microdomains leading to the generation of biologically functional membrane domains.     

Discrete logic modelling as a means to link protein signalling networks with functional analysis of mammalian signal transduction

Large‐scale protein signalling networks are useful for exploring complex biochemical pathways but do not reveal how pathways respond to specific stimuli. Such specificity is critical for understanding disease and designing drugs. Here we describe a computational approach—implemented in the free CNO software—for turning signalling networks into logical models and calibrating the models against experimental data. When a literature‐derived network of 82 proteins covering the immediate‐early responses of human cells to seven cytokines was modelled, we found that training against experimental data dramatically increased predictive power, despite the crudeness of Boolean approximations, while significantly reducing the number of interactions. Thus, many interactions in literature‐derived networks do not appear to be functional in the liver cells from which we collected our data. At the same time, CNO identified several new interactions that improved the match of model to data. Although missing from the starting network, these interactions have literature support. Our approach, therefore, represents a means to generate predictive, cell‐type‐specific models of mammalian signalling from generic protein signalling networks.     

Understanding signal transduction through functional proteomics

The study of signal transduction provides fundamental information regarding the regulation of all biologic processes that support the normal function of life. Functional proteomics, a rapidly emerging discipline that aims to understand the expression, function and regulation of the entire set of proteins in a given cell type, tissue or organism, offers unprecedented opportunity for signal transduction research in terms of understanding cellular behavior and regulation at the systems level. Indeed, swift progress in the area of proteomics has demonstrated the major impact of proteomic approaches on signal transduction and biomedical research. In this review, recent and innovative applications of functional proteomics in determining changes in protein contents, modifications, activities and interactions underpinning signaling transduction pathways are discussed.     

Biological implications of SNPs in signal peptide domains of human proteins

Proteins destined for secretion or membrane compartments possess signal peptides for insertion into the membrane. The signal peptide is therefore critical for localization and function of cell surface receptors and ligands that mediate cell-cell communication. About 4% of all human proteins listed in UniProt database have signal peptide domains in their N terminals. A comprehensive literature survey was performed to retrieve functional and disease associated genetic variants in the signal peptide domains of human proteins. In 21 human proteins we have identified 26 disease associated mutations within their signal peptide domains, 14 mutations of which have been experimentally shown to impair the signal peptide function and thus influence protein transportation. We took advantage of SignalP 3.0 predictions to characterize the signal peptide prediction score differences between the mutant and the wild-type alleles of each mutation, as well as 189 previously uncharacterized single nucleotide polymorphisms (SNPs) found to be located in the signal peptide domains of 165 human proteins. Comparisons of signal peptide prediction outcomes of mutations and SNPs, have implicated SNPs potentially impacting the signal peptide function, and thus the cellular localization of the human proteins. The majority of the top candidate proteins represented membrane and secreted proteins that are associated with molecular transport, cell signaling and cell to cell interaction processes of the cell. This is the first study that systematically characterizes genetic variation occurring in the signal peptides of all human proteins. This study represents a useful strategy for prioritization of SNPs occurring within the signal peptide domains of human proteins. Functional evaluation of candidates identified herein may reveal effects on major cellular processes including immune cell function, cell recognition and adhesion, and signal transduction.     

Phosphoinositide-binding domains: Functional units for temporal and spatial regulation of intracellular signalling

Inositol phospholipid (phosphoinositide) is a versatile lipid characterized by its isomer-specific localization, as well as its molecular diversity attributable to phosphorylation events. Phosphoinositides act as signal mediators in a spatially and temporally controlled manner. Information about the timing and location of their production is received by phosphoinositide-binding proteins and transmitted to multiple lines of intracellular events such as signal transduction, cytoskeletal rearrangement, and membrane trafficking. Among those proteins, a significant portion possess globular structural units, called domains, which are specialized for phosphoinositide binding. The pleckstrin homology (PH) domain was the first phosphoinositide-binding domain identified. It contains the largest number of members and is associated with the formation of signalling complexes on the plasma membrane. Recent studies identified other novel phosphoinositide-binding domains (Fab1p, YOTB, Vps27p, EEA1 (FYVE), Phox homology (PX), and epsin N-terminal homology (ENTH)), thus extending our knowledge of how the functional versatility of phosphoinositides is achieved.     

A Novel Assay for Assessing Juxtamembrane and Transmembrane Domain Interactions Important for Receptor Heterodimerization

Understanding the basis of specificity in receptor homodimerization versus heterodimerization is essential in determining the role receptor plays in signal transduction. Specificity in each of the interfaces formed during signal transduction involves cooperative interactions between receptor extracellular, transmembrane (TM), and cytoplasmic domains. While methods exist for studying receptor heterodimerization in cell membranes, they are limited to either TM domains expressed in an inverted orientation or capture only heterodimerization in a single assay. To address this limitation, we have developed an assay (DN-AraTM) that enables simultaneous measurement of homodimerization and heterodimerization of type I receptor domains in their native orientation, including both soluble and TM domains. Using integrin α_IIb and RAGE (receptor for advanced glycation end products) as model type I receptor systems, we demonstrate both specificity and sensitivity of our approach, which will provide a novel tool to identify specific domain interactions that are important in regulating signal transduction.     

Properties of an Ezrin Mutant Defective in F-actin Binding

Ezrin, radixin and moesin are a family of proteins that provide a link between the plasma membrane and the cortical actin cytoskeleton. The regulated targeting of ezrin to the plasma membrane and its association with cortical F-actin are more than likely functions necessary for a number of cellular processes, such as cell adhesion, motility, morphogenesis and cell signalling. The interaction with F-actin was originally mapped to the last 34 residues of ezrin, which correspond to the last three helices (αB, αC and αD) of the C-terminal tail. We set out to identify and mutate the ezrin/F-actin binding site in order to pinpoint the role of F-actin interaction in morphological processes as well as signal transduction. We report here the generation of an ezrin mutant defective in F-actin binding. We identified four actin-binding residues, T576, K577, R579 and I580, that form a contiguous patch on the surface of the last helix, αD. Interestingly, mutagenesis of R579 also eliminated the interaction of band four-point one, ezrin, radixin, moesin homology domains (FERM) and the C-terminal tail domain, identifying a hotspot of the FERM/tail interaction. In vivo expression of the ezrin mutant defective in F-actin binding and FERM/tail interaction (R579A) altered the normal cell surface structure dramatically and inhibited cell migration. Further, we showed that ezrin/F-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway. Taken together, these observations highlight the importance of ezrin/F-actin function in the development of dynamic membrane/actin structures critical for cell shape and motility, as well as signal transduction.